Ab18766

Peroxisome proliferator-activated receptor alpha (PPARA) and CASP12 were localised by immunohistochemistry in mouse placental tissue derived from both normal (nZ10) and obese (nZ10) fathers. In brief, paraffin sections (5 mm) were dewaxed in xylene and rehydrated through descending grades of ethanol (unless otherwise stated, all chemicals were obtained from Sigma-Aldrich). Sections underwent antigen retrieval using 0.01 mol/l sodium citrate buffer (pH 6.0) for 15 min and then incubation in the hot buffer for a further 20 min. Sections were washed for 5 min in PBS, pH 7.6. Following endogenous peroxidase quenching and blocking of non-specific binding, sections were incubated overnight at 4 8C with rabbit anti-PPARA (ab8934, Abcam, Cambridge, UK) at a concentration of 5 mg/ml in PBS or rabbit anti-caspase-12 (CASP12; ab18766, Abcam) at a concentration of 1 mg/ml in PBS. For isotype controls, primary antibody was substituted with rabbit IgG. Staining was visualised using the SuperPicture Kit (Invitrogen) followed by peroxidase substrate 3,3 0 -diaminobenzidine (DAB) (Dako, Glostrup, Denmark), and lightly counterstained with Harris hematoxylin. Sections were then dehydrated and mounted. Staining was visualised and captured using a Leica microscope and camera.

Right hemispheres were homogenized and separated by SDS-PAGE according to the Laemmli method and transferred to nitrocellulose membranes. Immunolabeling was performed using antibodies against Sigma-1 receptor (ab53852; 1:500; Abcam, Cambridge, UK), DDIT3 [EPR4943(2)]-C-terminus (ab179823; 1:1,000; Abcam), caspase-12 (ab18766; 1:1,000; Abcam), and active caspase-3 (Beyotime). Sig-1R, C/EBP homologous protein (CHOP), caspase-12, and caspase-3 levels were normalized using anti-glyceraldehyde-3-phosphate dehydrogenase as a loading control to confirm equal amounts of protein. Horseradish peroxidase (HRP)conjugated goat anti-rabbit antibody (Boster Biological Technology, Wuhan, China) and HRP-conjugated goat anti-mouse antibody (Boster Biological Technology) were used as the secondary antibodies. Immunoreactivity was quantified using a chemiluminescence kit (Beyotime) and exposure to film. Quantitative analysis of band intensity was carried out using the ImageJ Software (National Institutes of Health, Bethesda, Md).